Although tree-thinking skills are crucial for understanding biology, research on this topic remains scarce in Asian countries. This study assessed tree-thinking skills among first-year university students in Japan using BETTSI (Basic Evolutionary Tree-Thinking Skills Instrument 3.0). Additionally, it analyzed the presentation of evolutionary diagrams in the biology textbooks used by these students during high school. The results showed that while students with high school advanced biology had more experience and confidence with phylogenies, their overall BETTSI scores did not differ. Furthermore, Japanese university students’ overall BETTSI scores were comparable to those reported in previous studies of American university students majoring in STEM fields. Item-by-item analysis revealed that Japanese university students face challenges concerning interpreting phylogenetic relatedness and comparing phylogenies compared to their American counterparts. The textbook analysis suggests that the instruction aimed at developing these skills is likely not being provided.